This is a continuing proposal to study the mechanism and regulation of ureagenesis and its inherited disorders. Our previous studies have elucidated the correlations between sequence, structure and function of ornithine transcarbamylase (OTC) and the spectrum of mutation and genotype/phenotype relationships. A yeast model of the human ornithine transporter has been created and the wild type and prevalent mutant characterized in the mitochondrial environment. The mouse and human N-acetylglutamate synthase (NAGS) genes were identified and cloned and mutations causing hyperammonemia were identified. Recently, we found evidence for multiple NAGS proteins in the liver and for in vivo and in vitro interactions between NAGS and carbamyl phosphate synthetase I (CPSI). We also showed that N-carbamylglutamate (NCG) restores ureagenesis to normal in NAGS deficient patients. This proposal focuses on the mechanisms by which ureagenesis is regulated by NAGS protein processing, NAGS-CPS interactions and N- acetylglutamate's role in modulating nitrogen flux through the urea cycle. The specific aims are.to: 1. Investigate functional and structural changes in NAGS proteins of liver and intestine in response to changing nitrogen load. 2. Characterize the interactions between NAGS and CPSI. 3. Study the effect of the NAG analog NCG on ureagenesis in healthy volunteers and patients with urea cycle disorders and organic acidemias using stable isotope enrichment methods. The N-termini of the variably processed NAGS proteins will be determined. The amounts of the different NAGS proteins and how they change in response to nitrogen intake will be determined using western blotting and mass-spectrometry. The interactions between CPSI and the "mature" and "conserved" NAGS proteins, as well as the variable domain peptide (VDP) will be characterized by immunoprecipitation, protein affinity chromatography, analytical ultracentrifugation, protein cross-linking and mass spectrometry, and the effects on catalysis will be studied by enzyme kinetics experiments. In a study involving healthy volunteers, in vivo, steady state, stable isotope enrichment methodology will be used to explore the role of NAG in short-term regulation of ureagenesis by measuring ureagenesis before and after NCG administration on low vs. high protein intake. The same methodology will then be used to examine the effect of NCG on ureagenesis in patients with primary NAG deficiency (NAGS deficiency), NAG dependency (CPSI deficiency) and secondary NAG deficiency (propionic, methylmalonic acidemia).